biotechnology: status and uses
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Biotechnology: Status and Uses. Animal Science 434 John J. Parrish. Reproductive Biotechnology's. Artificial insemination In vitro embryo production In vivo embryo production Embryo transfer Gender selection Stem Cells Genetic engineering Cloning. In Vitro Production of Embryos. - PowerPoint PPT PresentationTRANSCRIPT
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Biotechnology: Status and Uses
Biotechnology: Status and Uses
Animal Science 434
John J. Parrish
Animal Science 434
John J. Parrish
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Reproductive Biotechnology'sReproductive Biotechnology's
• Artificial insemination
• In vitro embryo production
• In vivo embryo production
• Embryo transfer
• Gender selection
• Stem Cells
• Genetic engineering
• Cloning
• Artificial insemination
• In vitro embryo production
• In vivo embryo production
• Embryo transfer
• Gender selection
• Stem Cells
• Genetic engineering
• Cloning
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In Vitro Production of EmbryosIn Vitro Production of Embryos
• Oocyte isolation and maturation
• Sperm preparation
• Sperm capacitation
• Fertilization
• Embryo Development
• Oocyte isolation and maturation
• Sperm preparation
• Sperm capacitation
• Fertilization
• Embryo Development
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In Vivo Embryo ProductionIn Vivo Embryo Production
• Normal cycling female– horses
• Superovulated female– Cattle– Sheep– Goats– Deer– Humans
• Normal cycling female– horses
• Superovulated female– Cattle– Sheep– Goats– Deer– Humans
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Stimulating Follicular DevelopmentStimulating Follicular Development
Est
rus
Est
rus
eCG orFSH
eCG orFSH
Progesterone From C.L.
Progesterone From C.L.
First FollicularWave
First FollicularWave
10-1210-12
MultipleOvulations
MultipleOvulations
PGF2PGF2
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Non-Surgical Embryo Flush
Non-Surgical Embryo Flush
Day 6 or 7Day 6 or 7
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Stage of Embryo at Recovery Stage of Embryo at Recovery
Tight Morula (day 5 - 7)Tight Morula (day 5 - 7)
Early Blastocyst (day 7 - 8)Early Blastocyst (day 7 - 8)
Blastocyst (day 7 - 9)Blastocyst (day 7 - 9)
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ET - Scheme
ET - Scheme
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Embryo SexingEmbryo Sexing
• Hy Antigen– Associated with male cells
• PCR and Detection of Y and X DNA
• Hy Antigen– Associated with male cells
• PCR and Detection of Y and X DNA
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Hy-AntigenHy-Antigen
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Octopus Springs - Yellowstone National ParkOctopus Springs - Yellowstone National Park
Home of Thermus aquaticus - Taq PolymeraseHome of Thermus aquaticus - Taq Polymerase
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PCR Approach to Embryo SexingPCR Approach to Embryo Sexing
XY
F M F F F F F F
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Sperm Gender SelectionSperm Gender Selection
Selection of X or Y spermSelection of X or Y sperm
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PERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOME
PERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOME
Human 2.9
Cattle 3.8
Chinchilla 7.5
Turkey 0
Human 2.9
Cattle 3.8
Chinchilla 7.5
Turkey 0
X Chromosome has more DNA!!!X Chromosome has more DNA!!!
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++ --+-
LLAASSEERR
90°0°
XX ssoorrttYY ssoorrtt
SpermStained
With DNASensitive
FluorescentDye
SpermStained
With DNASensitive
FluorescentDye
Flow Cytometer Separation of X and Y SpermFlow Cytometer Separation of X and Y Sperm
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Sperm Gender SelectionSperm Gender Selection
• Flow Cytometry– Only method that works!– Very few sperm recovered– Reduced fertility– Expense will limit use
• Flow Cytometry– Only method that works!– Very few sperm recovered– Reduced fertility– Expense will limit use
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Fetal SexingFetal Sexing
OR
Ultrasound EvaluationUltrasound Evaluation• Day 55 - 65• Day 55 - 65
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CloningCloning
Split morulaSplit morula
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Cloning by Nuclear TransferCloning by Nuclear Transfer
Cycles are limited
Only 3 - 4 cycles
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Nuclear Cloning to Produce Stem CellsNuclear Cloning to Produce Stem Cells
Cells of InterestGrown in Culture
Dish
Cells of InterestGrown in Culture
Dish
Mature Oocyte enucleated and cell
of interest fused
Mature Oocyte enucleated and cell
of interest fused
Embyro??Develops
Embyro??Develops
Inner Cell Mass Cells isolated and grown in culture
Inner Cell Mass Cells isolated and grown in culture
DifferentiateDifferentiatePotential Tissue:• Neural• Adipose• Connective• Heart
Potential Tissue:• Neural• Adipose• Connective• Heart
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Stem Cells Grown in Culture and Allowed to Differentiate
Stem Cells Grown in Culture and Allowed to Differentiate
http://www.ansci.wisc.edu/jjp1/as434/powerpoint/fa07/stem_cells.mov
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Benefits of Stem Cell Research
Benefits of Stem Cell Research
1. Regenerative medicine and tissue engineering
In vitro systems: drug discovery, toxicology, diagnostic assays and cell culture reagents
Biomedical research: unravel mechanisms of disease and human development
1. Regenerative medicine and tissue engineering
In vitro systems: drug discovery, toxicology, diagnostic assays and cell culture reagents
Biomedical research: unravel mechanisms of disease and human development
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Genetic Manipulation
Genetic Manipulation
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Transgenic for Growth Hormone
Transgenic for Growth Hormone
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Gene Transfer Using Micro-Injection of Pronuclei
Gene Transfer Using Micro-Injection of Pronuclei
Less than 1% efficiency
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Gene Transfer
Using Viral Transfection
Gene Transfer
Using Viral Transfection
Better success but left with potential for viral replicationBetter success but left with potential for viral replication